A agropecuária brasileira apresenta relevante papel sócio-econômico para o país, e constantemente busca novas tecnologias para alcançar uma agricultura sustentável. Com as mudanças que vêm ocorrendo no uso da terra, principalmente nas regiões tropical e subtropical, o Brasil vem sendo apontado como um grande emissor dos gases do efeito estufa. A conversão de florestas em sistemas agrícolas pode levar a um rápido aumento dos fluxos de CO2, CH4 e N2O no ambiente, além de potencializar o efeito estufa e ameaçar os diferentes ecossistemas. Em busca de sistemas mais conservacionistas, que possam mitigar o efeito estufa, os sistemas convencional, plantio direto, integração lavoura-pecuária e pastagem com histórico bem definido, foram selecionados nesse estudo para melhor compreensão e discernimento das possíveis mudanças oriundas dos sistemas avaliados no bioma do Cerrado. Em um segundo momento avaliamos o potencial da elevada concentração de CO2 aquecimento das parcelas em sistema sob temperature freeair controlled enhancement e carbon dioxide free-air enrichment (T-FACE) para avaliar as alterações funcionais e composição microbiana do solo. Os objetivos desse estudo foram: determinar a quantidade de células total dos genes 16S rRNA bactéria, archaea e dos genes funcionais amoA, nirS, nirK, cnorB, nosZ, presentes em diferentes sistemas de manejo do solo. Bem como, possíveis alterações na comunidade microbiana do solo sob elevada concentração de CO2 e aquecimento das parcelas. Para acessar o número de cópias dos genes foi utilizado o PCR quantitativo, a estrutura da comunidade microbiana foi determinada pela técnica de T-RFLP e a composição microbiana pelo sequenciamento de terceira geração. Os resultados dos sistemas de plantio direto e integração lavoura-pecuária revelaram importante capacidade de controlarem as emissões de N2O. Notoriamente, o número de cópias do gene nosZ teve sua densidade incrementada nos dois sistemas de plantio direto e integração lavoura-pecuária, este gene apresenta alto potencial para monitorar a desnitrificaçnao completa do N2O a N2. Adicionalmente, a elevada concentração de CO2 e elevada temperatura incrementaram o número de cópias dos genes nifH, AOB e nosZ ao longo do experimento. A análise da diversidade dos grupos taxinômicos e funcional revelou que a diversidade funcional foi alterada nas parcelas com maior emissão de N2O, apresentando maior abundância de genes (2-3 vezes) envolvidos na desnitrificação, acarretando possivelmente essas maiores emissões de N2O pela microbiota do solo. / Agriculture activities have large an important socio-economic role for a country, and are constantly searching for new technologies to achieve sustainable agriculture. Changes have occurred in land use, especially in tropical and subtropical regions and Brazil has been considered as a large emitter of greenhouse gases from agricultural systems. The conversion of forests to agricultural systems can lead to a fast increase of CO2 streams, CH4 and N2O for atmosphere, which enhances the greenhouse effect and threaten the ecosystem. In search of more conservation systems that can mitigate the greenhouse gas, the conventional, no-tillage, integrated crop-livestock and pasture systems with well defined historical management were selected in this study to better understand and decifer the possibles changes resulting in the biome Cerrado. In a second study, it was evaluated the potential of high concentration of CO2 and warming plots on system under increased temperature free-air controlled enhancement e carbon dioxide freeair enrichment (T-FACE) to assess the functional changes and microbial composition in the soil. The objectives of this study were to determine the total amount of the 16S rRNA Bacteria, Archaea and the functional genes amoA, nirS, nirK, cnorB, nosZ present under different soil management and evaluate the possible changes in the soil microbial community under high CO2 concentration and warming in the plots. To access the number of copies genes we used quantitative PCR, with the microbial community structure determined by T-RFLP and the microbial composition by Illumina next-generation sequencing. No-tillage and integrated crop-livestock revealed important capability to control N2O emissions. Notably, the high number of nosZ gene copies was found under no-tillage and integrated crop-livestock systems. This gene has a high potential to monitor the oxidation of N2O to N2. In addition, high CO2 concentration and elevated temperature increased 2-3 folds the number of copies of the nifH genes, and AOB nosZ throughout the experiment. The analysis of the diversity of functional taxonomic groups revealed that functional diversity has changed in plots with high N2O emissions, and showed a greater abundance of genes involved in denitrification, which possibly has stimulated the emissions of N2O from soil microbiota.

A agropecuária brasileira apresenta relevante papel sócio-econômico para o país, e constantemente busca novas tecnologias para alcançar uma agricultura sustentável. Com as mudanças que vêm ocorrendo no uso da terra, principalmente nas regiões tropical e subtropical, o Brasil vem sendo apontado como um grande emissor dos gases do efeito estufa. A conversão de florestas em sistemas agrícolas pode levar a um rápido aumento dos fluxos de CO2, CH4 e N2O no ambiente, além de potencializar o efeito estufa e ameaçar os diferentes ecossistemas. Em busca de sistemas mais conservacionistas, que possam mitigar o efeito estufa, os sistemas convencional, plantio direto, integração lavoura-pecuária e pastagem com histórico bem definido, foram selecionados nesse estudo para melhor compreensão e discernimento das possíveis mudanças oriundas dos sistemas avaliados no bioma do Cerrado. Em um segundo momento avaliamos o potencial da elevada concentração de CO2 aquecimento das parcelas em sistema sob temperature freeair controlled enhancement e carbon dioxide free-air enrichment (T-FACE) para avaliar as alterações funcionais e composição microbiana do solo. Os objetivos desse estudo foram: determinar a quantidade de células total dos genes 16S rRNA bactéria, archaea e dos genes funcionais amoA, nirS, nirK, cnorB, nosZ, presentes em diferentes sistemas de manejo do solo. Bem como, possíveis alterações na comunidade microbiana do solo sob elevada concentração de CO2 e aquecimento das parcelas. Para acessar o número de cópias dos genes foi utilizado o PCR quantitativo, a estrutura da comunidade microbiana foi determinada pela técnica de T-RFLP e a composição microbiana pelo sequenciamento de terceira geração. Os resultados dos sistemas de plantio direto e integração lavoura-pecuária revelaram importante capacidade de controlarem as emissões de N2O. Notoriamente, o número de cópias do gene nosZ teve sua densidade incrementada nos dois sistemas de plantio direto e integração lavoura-pecuária, este gene apresenta alto potencial para monitorar a desnitrificaçnao completa do N2O a N2. Adicionalmente, a elevada concentração de CO2 e elevada temperatura incrementaram o número de cópias dos genes nifH, AOB e nosZ ao longo do experimento. A análise da diversidade dos grupos taxinômicos e funcional revelou que a diversidade funcional foi alterada nas parcelas com maior emissão de N2O, apresentando maior abundância de genes (2-3 vezes) envolvidos na desnitrificação, acarretando possivelmente essas maiores emissões de N2O pela microbiota do solo. / Agriculture activities have large an important socio-economic role for a country, and are constantly searching for new technologies to achieve sustainable agriculture. Changes have occurred in land use, especially in tropical and subtropical regions and Brazil has been considered as a large emitter of greenhouse gases from agricultural systems. The conversion of forests to agricultural systems can lead to a fast increase of CO2 streams, CH4 and N2O for atmosphere, which enhances the greenhouse effect and threaten the ecosystem. In search of more conservation systems that can mitigate the greenhouse gas, the conventional, no-tillage, integrated crop-livestock and pasture systems with well defined historical management were selected in this study to better understand and decifer the possibles changes resulting in the biome Cerrado. In a second study, it was evaluated the potential of high concentration of CO2 and warming plots on system under increased temperature free-air controlled enhancement e carbon dioxide freeair enrichment (T-FACE) to assess the functional changes and microbial composition in the soil. The objectives of this study were to determine the total amount of the 16S rRNA Bacteria, Archaea and the functional genes amoA, nirS, nirK, cnorB, nosZ present under different soil management and evaluate the possible changes in the soil microbial community under high CO2 concentration and warming in the plots. To access the number of copies genes we used quantitative PCR, with the microbial community structure determined by T-RFLP and the microbial composition by Illumina next-generation sequencing. No-tillage and integrated crop-livestock revealed important capability to control N2O emissions. Notably, the high number of nosZ gene copies was found under no-tillage and integrated crop-livestock systems. This gene has a high potential to monitor the oxidation of N2O to N2. In addition, high CO2 concentration and elevated temperature increased 2-3 folds the number of copies of the nifH genes, and AOB nosZ throughout the experiment. The analysis of the diversity of functional taxonomic groups revealed that functional diversity has changed in plots with high N2O emissions, and showed a greater abundance of genes involved in denitrification, which possibly has stimulated the emissions of N2O from soil microbiota.

Le protoxyde d’azote (N2O) est un gaz à effet de serre (GES) important et la principale substance attaquant la couche d'ozone. Les sols agricoles sont la principale source anthropique de ce GES. La concentration de N2O dans l'atmosphère est en constante augmentation, mais nous manquons de connaissances sur les facteurs contrôlant sa production et sa consommation dans les sols. La réduction du N2O en N2 par des microorganismes porteurs du gène codant pour la N2O réductase (nosZ) est le seul processus biologique capable de réduire ce GES. Des études récentes ont mis en évidence un clade précédemment inconnu de réducteurs du N2O qui interfère de manière significative avec la quantité de N2O produite dans les sols. Cette thèse a cherché à mieux comprendre l'écologie des réducteurs du N2O dans les sols agricoles.Une combinaison d'expériences d'incubation en laboratoire mais aussi d’expériences en plein champs a été utilisée pour essayer de mieux comprendre la production de N2O dans le sol, en analysant l’influence conjointe des producteurs et réducteurs de N2O. Nous avons aussi évalué l’impact des pratiques agricoles et leurs potentiels à modifier ces communautés microbiennes. Suite aux essais réalisés en laboratoire, nous avons montré que l'ajout d'une souche non-dénitrifiante Dyadobacter fermentans,possédant la N2O réductase NosZII, permettait de réduire la production de N2O dans 1/3 des sols testés. Certains sols sont même devenus consommateurs de N2O suite à l'ajout de la souche nosZII. Cette expérience a démontré la contribution des bactéries nosZII non-dénitrifiantes dans la consommation de N2O dans le sol.D’autre part, nos analyses en contexte agricole ont montré que les pratiques agricoles testées ont peu d’influence sur les communautés microbiennes considérées, les exceptions étant le travail du sol (labour), et le système de culture (annuel ou pérenne). L’intensifiant du travail du sol induit une augmentation de la diversité de nosZII. Nous observons le même phénomène dans le système de culture annuel comparé au système de culture pérenne. D’autres résultats nous permettent aussi d’affirmer que le clade récemment identifié de réducteurs du N2O est plus sensible aux variables environnementales que le clade précédemment connu (nosZI). Les variations de propriétés du sol, notamment pH et C:N structurent les communautés microbiennes appartenant à ces 2 clades indiquant une spécialisation de niche pour chacun de ces deux clades de N2O-réducteurs.Pour mieux comprendre les relations entre les communautés microbiennes et les processus impliqués, nous avons évalué les activités potentielles de dénitrification et de nitrification, et les émissions de N2O in situ. La production potentielle de N2O et l'activité potentielle de dénitrification ont été utilisées pour calculer le ratio de production de N2O (N2O:N2). La diversité du clade nosZII est négativement corrélée au ratio N2O:N2, et explique à elle seule la plus grande part de variance observée du ratio N2O:N2. Les variations de production potentielle de N2O et d'activité potentielle de dénitrification sont elles expliquées principalement par les variations de propriétés du sol. Afin de mieux évaluer la contribution des différents facteurs édaphiques et microbiologiques aux variations d’émission in situ de N2O, 70000 mesures ont été subdivisées en différentes gammes d’émission de N2O, d‘émissions dites de base à des émissions élevées. Fait intéressant, les variations d’émissions in situ de N2O dites de base sont seulement liées à des variations du pH du sol, alors que les variations d’émissions dites élevées sont également fortement associées aux variations de diversité des communautés microbiennes. Parmi les variables microbiennes importantes, nous avons constaté que la diversité des nosZII est négativement liée aux émissions de N2O in situ dites élevées.En conclusion, nos résultats mettent en évidence l’importance du clade nosZII pour le cycle du N2O dans le sol (...). / Nitrous oxide (N2O) is an important greenhouse gas (GHG) and the main ozone depleting substance. Agricultural soils are the main anthropogenic-induced source of this GHG. The concentration of N2O in the atmosphere is steadily increasing, but we still lack knowledge on the factors controlling its production and consumption in soils. The reduction of N2O to N2 by microorganisms harboring the N2O reductase gene (nosZ) is the only known biological process able to consume this GHG. Recent studies revealed a previously unknown clade of N2O-reducers which was shown to be important to the N2O sink capacity of soils. This thesis seeks to gain a greater understanding on the ecology of N2O-reducers in agricultural soils. A combination of laboratory incubation and field experiments were used to gain knowledge on the importance of N2O-producers and N2O-reducers to the soil N2O production. Additionally, the potential of agricultural practices to modify those microbial communities were assessed.We showed experimentally, in laboratory incubations, that the addition of a non-denitrifying strain Dyadobacter fermentans, which possesses the previously unaccounted N2O reductase NosZII, reduced N2O production in 1/3 of the tested soils. Remarkably, after addition of the nosZII strain, some soils became a N2O sink, as negative rates were recorded. This experiment provided unambiguous evidence that the overlooked non-denitrifying nosZII bacteria can contribute to N2O consumption in soil.Our evaluation of agricultural field experiments showed limited impact of agricultural practices on the microbial communities except for tillage management, and differences observed between an annual and a perennial cropping system. Increasing tillage management enhanced nosZII diversity. Higher diversity of the nosZII clade was also observed in the annual cropping system than in the perennial cropping system. Overall, the recently identified clade of N2O-reducers was more sensitive to environmental variables than the previously known clade (nosZI). The community structure of these two groups was explained by common and uncommon soil properties suggesting niche specialization between the two N2O-reducers.In an attempt to understand the relationship between the microbial communities and process rates, we assessed the potential denitrification and nitrification rates, and in situ N2O emissions. Potential N2O production and potential denitrification activity were used to calculate the denitrification end-product ratio. The diversity of nosZII was negatively related to the N2O:N2 ratio and explained the highest fraction of its variation (26%), while the potential N2O production and potential denitrification activity were mainly explained by the soil properties. To better evaluate the contribution of different factors to the in situ emissions, more than 70000 N2O measurements were subdivided into different ranges, from low to high rates. Interestingly, the low range of in situ N2O emissions was only related to soil pH, while the high ranges were also strongly related to the microbial communities. This result suggests that the “base-line” N2O emissions might be more regulated by soil edaphic conditions than by microorganisms, the lasts being more important for the high emissions ranges. Among the significant microbial variables, we found that the diversity of nosZII was negatively related to the high ranges of in situ N2O emissions.In conclusion, our results highlight the relevance of the second clade of N2O-reducers to the fate of N2O in soil. Our results also suggest niche differentiation between the two N2O-reducing clades with nosZII being more responsive to environmental variables. Agricultural practices showed limited impact on the two guilds. Further research is needed to test the niche specialization between the two groups, to disentangle their controlling factors, and to evaluate their potential for N2O mitigation.

Bacteria play important roles in the transformation of nutrients in wetlands, but few studies have examined parameters affecting variation in bacterial community composition between wetlands. We compared the composition of eubacterial and denitrifying bacterial biofilm communities in 32 agricultural constructed wetlands in southern Sweden, and the extent to which wetland environmental parameters could explain the observed variation. Structure and richness of the eubacterial 16S rRNA gene and three denitrifying bacterial enzyme genes (nirK, nirS and nosZ), analysed by molecular fingerprinting methods, varied among the constructed wetlands, which could be partly explained by different environmental parameters. Results from the enzyme gene analyses were also compared to determine whether the practice of using a single denitrifying bacterial gene could characterize the overall community composition of denitrifying bacteria. We found that nirK was more diverse than both nirS and the nosZ, and the band structure and richness of the three genes were not related to the sam environmental parameters. This suggests that using a single enzyme gene may not suffice to characterize the community composition of denitrifying bacteria in constructed agricultural wetlands. / <p>Included in doctoral thesis: Milenkovski, Susann. Structure and Function of Microbial Communities in Constructed Wetlands - Influence of environmental parameters and pesticides on denitrifying bacteria. Lund University 2009.</p>

Nitrogen cycling processes can be tracked using quantitative Polymerase Chain Reaction (qPCR) to determine the presence and qReverse Transcriptase-PCR (qRT-PCR) to determine expression of key genes, or ‘biological markers’, for nitrogen metabolism. Nitrification is catalyzed in part, by two enzymes: ammonia monooxygenase (AMO; NH3 NH2OH) and nitrite oxidoreductase (NXR; NO2- NO3-). For denitrification, four enzymes act sequentially: nitrate reductase (NAR/NAP; NO3- NO2-), nitrite reductase (NIR; NO2- NO), nitric oxide reductase (NOR; NO  N2O), and nitrous oxide reductase (NOS; N2O  N2). A principle of wastewater treatment (WWT) is to remove excess nitrogen by taking advantage of natural nitrogen cycling or biological nitrogen removal (BNR). This process involves using microorganisms to bring influent ammonia through nitrification and denitrification to release nitrogen gas, which does not contribute to eutrophication. A novel shortcut nitrogen removal configuration could increase nitrogen removal efficiency by promoting nitritation/denitritation, reducing the classic nitrogen cycle by removing the redundant oxidation/reduction step to nitrate (NO3-). Here, three nitrogen transformations were used to track the three main phases in the nitrogen cycle; ammonia monooxygenase for nitrification, nitrite oxidoreductase for shortcut, and nitrous oxide reductase for denitrification. Primers for qPCR and qRT-PCR were designed to capture as much sequence diversity as possible for each step. Genes from bacteria known to perform the nitrogen transformations of interest (amoA, nxrB, nosZ) were used to BLAST-query the Integrated Microbial Genomes & Microbiomes database (img.jgi.doe.gov) to find homologs from organisms commonly found in WWT. These sequences were then aligned to find regions sufficiently conserved for primer design. These PCR primers were tested against standards for each gene and used to track nitrogen transformation potential and expression in a novel lab-scale algal photo-sequencing batch reactor which promotes shortcut nitrogen removal from wastewater across three solids retention times (SRT, or mean cell residence time); 5, 10 and 15 days. SRT 15 had the greatest total nitrogen removal with nitritation and denitritation observed. Nitrate was not detected in the first cycle and shortcut nitrogen removal was supported by low levels of nxrB genes and transcripts. Simultaneous nitrification/denitrification was supported by elevated concentrations of nosZ during the light period and less nitrite produced than ammonium consumed. Nitritation was predominantly performed by Betaproteobacteria amoA and nitrous oxide reduction was predominantly from nosZ group I (Proteobacteria-type).

Nitrous oxide (N2O) is a greenhouse gas (GHG) with 300 times the radiative forcing in the atmosphere of carbon dioxide (CO2), and has recently become a subject of great concern because the nitrogen (N) fertilizers which have been necessary to increase agricultural productivity have also dramatically increased N2O emissions from agroecosystems. Many N control practices have been suggested and implemented in agroecosystems, but their ability to simultaneously remove reactive N from the environment and prevent the production of N2O is, at best poorly understood. The goal of this work is to characterize environmental controls on production of N2O in denitrifying bioreactors. The review portion of this work first discusses the geologic history of the N cycle, how its past and present processes differ, and how it is being affected by human activity. It then explores the N cycle's biochemical pathways, reviews the controls for each of its steps, and discusses the environmental drivers of these controls. The review closes with a discussion of environmental N management strategies. The experimental portion of this work further explores these concepts by observing how biochar amendment and the modification of pH affect N2O production in the denitrification pathway in denitrifying bioreactors. Both pH and biochar have previously been shown to affect N2O production and many N management practices utilize biochar or manipulate pH to increase N retention. The objectives of the experiment were to: 1) Examine headspace N2O concentration in sealed, biochar-amended, denitrifying bioreactors; 2) Determine if the effects of pH on N2O production differ in biochar-amended systems versus controls (under acidic, unbuffered, and buffered conditions); 3) Quantify key denitrification genes (nirK, nirS, nosZ) in each treatment combination. Experimental results showed biochar treatment to significantly increase N2O emissions, a result which runs contrary to most, but not all studies regarding its effects on N2O production. Differences between treatments decreased with increasing pH levels. Biochar did not exhibit significant effects on individual denitrification genes, but it did show influence on the ratios of their populations. On the other hand, pH was found to have significant effects on nirS and nosZ populations. Differences in N2O production between biochar and controls were thus explained by biochar's chemical effects, likely its ability to increase denitrification activity. Developing an understanding of the mechanisms behind these differences will require using a combination of isotope tracing, enzyme assays, and mass balance approaches. Future microbial work in biochar-amended systems should attempt to characterize differences in gene expression, overall community structure, and long-term population trends in the genes of interest. The combination of these approaches should allow researchers to better predict where N2O production will occur and develop strategies to mitigate it while simultaneously increasing food production to meet the demands of a growing population. / Master of Science

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